Project Summary: The machine used for template-guided protein synthesis?the ribosome and its associated translation factors?has been exploited for over 20 years to site-specifically incorporate > 150 unnatural ?-amino acids and > 10 ?-hydroxy acids into proteins, in vitro, in cells, and in animals. More exotic monomers, including N-alkyl ?-amino acids, D-amino acids, and some ?-amino acids have been incorporated using cell-free in vitro systems. These efforts have deepened our understanding of protein and RNA function and provided real-world commodities such as antibody-drug conjugates, modified therapeutics, and biomaterials to benefit society. Yet, even after two-plus decades of research, until very recently?the preliminary results for this application?there were no reports of a ribosome able to introduce a ?-amino acid into a protein in vivo, nor any sophisticated bioinformatics tools for ribosome redesign. The goal of this application is to develop and apply genetic engineering, bioinformatics, and chemical biology tools to repurpose the bacterial translational apparatus for the templated, in vivo biosynthesis of proteins and polypeptides containing backbone-modified monomers. The primary focus is on ?- amino acids, as preliminary results indicate that this goal is attainable with a 4-5 year RO1 award, but the strategies, insights, and tools developed will provide a robust infrastructure for the sequence-templated biosynthesis of diverse classes of evolvable matter?truly exotic biopolymers?whose functions are limited only by our collective imagination. By creating new machines that efficiently incorporate ?-amino acids into proteins in vivo, we will not only provide tools to address enduring questions about natural translation, but also provide real benefit to diverse segments of bio-medical research engine.